PROJECT SUMMARY Cancer encompasses a collection of diseases characterized by over-proliferation of abnormal cells. The disease can afflict all tissues and almost every cell type in the body, and decades of research have catalogued more than 28,000 genes harboring over 2 million unique mutations in human cancer cells. Which of these mutations are causal, which are consequential, and which are simply bystanders is a matter of continued debate. Adding to the confusion and uncertainty are often contradictory findings for the roles of bona fide oncogenes and tumor-suppressors in common cancers. Rather than a disease of individual proteins, cancer is a complex disease that is more likely to be understood as the malfunctioning of signaling pathways and networks. Indeed, mutations affecting the behavior of important signaling hubs, such as Ras/PKA, p53, and mTOR, and their associated signaling networks, are found overrepresented in many cancers. Understanding the architecture of the cell's signaling pathways, identifying the information these pathways transmit, and discovering how and where this information is processed to inform the decisions cells make regarding growth are the key to making sense of complex diseases such as cancer. Mapping the basic structure of the cell's circuitry has already begun but still largely awaits a functional data overlay. This grant proposal seeks to: 1) Dissect the circuitry lying upstream of TSC by comprehensively mapping regulatory interactions under a variety of relevant physiological conditions. 2) Relate identified regulatory sites to modes of TSC regulation, such as changes in localization, Rheb-GAP activity, and complex stability. 3) Establish the hierarchy of input signals by interrogating complex environments, those with multiple simultaneous stimuli. 4) This new mechanistic understanding of TSC regulation will be combined with identified patient mutations in TSC to gain deeper insight into the disease process and predict therapeutic oppertunities. 5) Identify and sensitize cancer cell lines with TSC dependent acquired therapeutic resistance. Together, this information will significantly aid the development of effective targeted therapies against TSC tumor syndromes and cancer.